免疫性和酒精性肝纤维化大鼠肝脏细胞质膜的蛋白质组学研究

目的 研究肝纤维化的发生机制,寻找新的与肝纤维化相关的生物学标志物. 方法将48只大鼠分为乙醇组、免疫组和对照组,分别进行乙醇灌胃、猪血清注射与等渗盐水注射处理.采用James's网状染色法染色并检测处理后第2、4、6、8周大鼠肝脏的病理学变化.将处理后第2、4、6、8周的各组大鼠分别处死4只后取肝,并将肝脏组织作匀浆处理,通过2次蔗糖密度梯度离心获得细胞质膜组分,用Western blot法检测细胞质膜的纯度.提取肝脏细胞质膜蛋白质,通过双向凝胶电泳分析各个时期的大鼠肝脏细胞质膜蛋白质,差异蛋白质点在酶解后经戴安纳升级液相色谱Ultimate 3000串联布鲁克高容量离子阱质谱HCT进行鉴定.并对被鉴定的差异蛋白质进行功能和定位的分类分析. 结果大鼠肝脏细胞质膜得到了有效富集.双向凝胶电泳分析第2周和第8周的大鼠肝细胞质膜蛋白质,共找到87个差异蛋白质点,这些蛋白质点经过质谱鉴定后对应于30个非冗余蛋白质,包括膜联蛋白A2,细胞支架角蛋白8和18. 结论膜联蛋白A2、细胞支架角蛋白8和18等蛋白质可成为肝纤维化诊断的新标志物.

Subcellular proteome analysis of immune or alcohol induced rat liver fibrosis

Objective To study the mechanism of liver fibrogenesis and to find new non-invasive biomarkers. Method In this study, we used subcellular proteomic technology to study the plasma membrane proteins related to immune or alcohol induced liver fibrosis. Rat liver fibrosis models were induced by pig serum or alcohol injection. The liver fibrogenesis were detected by James's staining in the rat models after 2,4, 6 and 8 weeks of treatment. The liver plasma membrane (PM) of the 2- and 8-week treatment model rats were enriched by two-step sucrose density gradient centrifugation. The purity of PM was verified by western blotting, and the plasma membrane proteins were extracted and analyzed by 2 DE. The differentially expressed proteins were identified by LC-MS/MS. Cellular location and function of these identified differential protein were classified. Results Immune or alcohol induced liver fibrosis rat models were successfully established. Liver plasma membrane was significantly enriched after sucrose density ultracentrifugation treatment. 87 differential protein spots were find out by 2DE combined with LC-MS/MS from the liver plasma membrane proteins of the 2- and 8-week treatment rat models, which corresponded to 30 non-redundant proteins including annexin A2, keratin 8 and keratin 18. Conclusions A list of differentially expressed proteins relate to liver fibrosis were successfully identified.Differential proteins such as annexin A2,keratin 8 and keratin 18 could be new biomarkers for liver fibrosis diagnosis.